Project Summary In the past decade, advances in next-generation sequencing technologies have led to an explosion of genomic studies of the human gut microbiome. Dysbiosis, or perturbation of the microbiome, has been linked to diseases such as inflammatory bowel disease and obesity; genetically modifying the gut microbiome thus holds promise as a method for treating these diseases. To achieve this goal, however, we must first develop a mechanistic understanding of the complex ecological dynamics of the microbiome. This requires the ability to make specific perturbations to the microbiome and observe the effects to determine causality, which we currently lack. There are no tools available to genetically manipulate most gut microbes, including at least 30% of species that are not cultivable in vitro. Engineering therapeutic functions into the microbiome also requires the ability to make targeted genomic edits, which presents a further challenge. In this project, I propose an in situ genome engineering system and a novel CRISPR-Transposon platform to address these key challenges. For in situ delivery of transposons into the gut microbiome, an E. coli donor strain will be orally gavaged into a live mouse host. The donor strain will conjugate a replicative or integrative plasmid vector into native gut microbes in situ to tag them with selectable markers such as GFP and antibiotic resistance genes. Using high- throughput screening methods and metagenomic sequencing of 16S rRNA, I will identify genetically engineerable microbes from fecal samples and determine the composition of reservoir populations for each plasmid vector. I will also track these populations over time to make metagenomic measurements of the dynamics of horizontal gene transfer (e.g. specific routes, time scales, and rates of transfer) in an animal-associated microbiome for the first time, which will elucidate this important aspect of gut microbial ecology. In this in situ system, integrative plasmids utilize a randomly inserting transposon to add new genetic functions into native microbes. Conversely, to enable targeted gene deletions and genetic knockout studies of diverse microbes, I will develop the CRISPR-Transposon (CRISPR-Tn) platform for targeted transposon mutagenesis. A fusion protein between a broad-host range transposase and a catalytically dead Cas9 endonuclease will be programmed to bind a specific genomic locus with by a synthetic guide RNA molecule. Once tethered, the transposase will be forced to insert transposons site-specifically. This programmable, host- independent system will enable targeted genome knockouts and operon insertions in a wide range of bacteria, and combined with in situ conjugative delivery, will significantly expand our genetic engineering capabilities for mechanistic studies of natural microbiomes.